Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST

Abstract

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multi-omic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1 ^-/- and NPC2 ^-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lyso-phosphatidylcholine species and multi-lamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS-complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2 ^-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

Keywords: Lysosome storage disorders; autophagy; cryo-ET; ferritinophagy; iNeuron; lipidomics; mitochondrial dysfunction; multi-omics; proteomics.

Figure 1.. Development and benchmarking of nMOST for proteomics and lipidomics analysis.

(A) Schematic of the nMOST method, which allows proteome and lipidome analysis by LC-MS. Lipid and protein extracts isolated from the same cell sources are sequentially injected onto LC prior to elution with an organic gradient and MS analysis (see STAR METHODS). (B) Chromatograms showing HEK293 cell peptide and lipid elution features during a 120 min gradient examining (left panel) total protein extract, (middle panel) total lipid extract, and (right panel) sequentially loaded protein and lipid extracts and nMOST analysis. The vast majority of peptides elute before 80 min while the majority of lipids elute between 80 and 120 min. (C) Peptide and lipid identifications from the corresponding LC-MS run in panel B. (D) Correlation of proteins (left panel) and lipids (right panel) identified by separate LC-MS (y-axis) versus nMOST (x-axis). r² values are >0.99. (E) Number of protein groups and lipid groups identified by nMOST versus μMOST methods. nMOST routinely out-performed μMOST for both proteins (left panel) and lipids (right panel). Amount of peptide injections are labelled above the line for each nMOST and μMOST. (F) Performance was comparable for both proteins and lipids when measured daily over a 7-day acquisition period. (G) nMOST allows simultaneous analysis of proteins and lipids from HEK293 cells, mouse brain extracts, C. elegans extracts, budding yeast extracts, human plasma, and lysosomes from HeLa cells isolated by Lyso-IP. (H) RSD values for the data in panel G.

Figure 2.. Landscape of total proteomes and lipidomes from LSD mutant cells using nMOST.

(A) Schematic describing the method for analysis of total cell extracts across 33 LSD mutants. Protein and lipid extracts were isolated from the samples in quadruplicate, and then sequentially injected for analysis by LC-MS over a 120 min gradient. (B,C) Panel B is a schematic depicting the method used for lipid/protein cross-correlation analysis employing a Kendall rank correlation (filtered for >1 association with Tau >0.4). Panel C shows a heatmap for Tau values. Clusters for proteins and lipids are shown. (D) Schematic showing the enrichment of specific lipids within individual lipid clusters. (E) Schematic showing the subset of GO term Cellular Compartment enriched within individual protein clusters. (F) Summed protein cluster 8 signature (sum abundance of all proteins within cluster 8 (enriched for autophagy terms) across the LSD mutant cells plotted as log₂FC (KO/WT). (G) Signature of protein cluster 5 (sum protein abundance relative to WT) across the LSD mutant cells.

Figure 3.. An nMOST-LSD Resource for Lipid-Protein Correlation Analysis.

(A) Schematic of search strategy to find functional protein-lipid relationships from LSD-nMOST cross-ome dataset. (B) Manhattan-style plot of lipid species (x-axis, lipid clusters 4, 7, 11) vs. protein correlations from protein cluster 8. Red dots represent proteins associated with GO-terms autophagy, autophagosome, lysosome, autolysosome. Additionally, select autophagy proteins are highlighted in viridis-color scheme. (C) Pie-chart of Top 10 enriched proteins from panel B. Autophagy receptors and autophagy core components represent ~50 of the hits and composition is shown on the right. (D) Screenshot of nMOST LSD on the Coon Lab Data Online portal. Tools available online are listed on the right. (E) Log₂FC ranked bargraph of indicated autophagy proteins across all analysed LSD genotypes. NPC1^−/− and NPC2^−/− genotypes are highlighted in shades of red. Data extracted from online portal. (F,G) Protein-lipid network extracted from protein cluster 8 and lipid clusters 4,7,11. F depicts protein-lipid connections for autophagy markers. G depicts protein-lipid connections for ferritinophagy markers. (H) Outlier analysis of two lipid species highlight correlated with NCOA4 and FTH1 (see G). Data extracted from online portal.

Figure 4.. Juxta-lysosomal accumulation of autophagy receptors and ferritin in NPC1^−/− and NPC2^−/− cells.

(A) Log₂FC relative to Control cells for the indicated autophagy receptors for 4KO cells. MAPLC3B: p(****) <0.0001, p(***) = 0.0001, p(*)=0.0129 & 0.0157. SQSTM1: p(****) <0.0001, p(**) = 0.0047. TAX1BP1: p(****) <0.0001, p(***) = 0.0001. NBR1: p(****) <0.0001. NCOA4: p(****) <0.0001, p(*) = 0.0244; FTH1: p(****) <0.0001; p(***) = 0.0007, p(**) = 0.0075. Data based on quadruplicate replicate nMOST measurements, ordinary one-way ANOVA with multiple comparisons, alpha = 0.05. Error bars depict S.D. (B) Western Blot for select autophagy and ferritinophagy proteins of whole cell lysates from HeLa Control treated for 3 days w/o U18666A. (C) The indicated cells were stained with Filipin to stain cholesterol-rich lysosomes and immunostained with α-LAMP1 and α-HA to detect TMEM192^−HA in lysosomes, followed by imaging with confocal microscopy. Scale bars = 10 μm. (D) Cells from panel C were imaged using 3D-SIM. Scale bar = 2 μm. (E) Immunostaining of Control and NPC2^−/− cells with α-LAMP1, α-LC3B and nuclei stained with DAPI. Line trace plots across individual Lamp1-positive lysosomes and the corresponding LC3B intensities. Scale bars = 5 μm and 2 μm (insets). Quantification of LC3B localization relative to LAMP1 is plotted on the right. (F) 3D-SIM reconstructions of HeLa Control cells treated for the indicated times with U18666A and NPC1^−/− immunostained for α-LAMP1 and α-panGABARAP, with lysosomes marked by Filipin staining. (G) Confocal fluorescent images of control and 4KO cells in Fed cells immunostained with α-FTH1 and α-HA to detect TMEM192^HA. Cholesterol-rich lysosomes were stained with Filipin and nuclei were stained with DNA SPY555. Scale bar = 20 μm. (H) Images from panel G were quantified by measuring Mander’s overlap between FTH1 signal and the lysosome mask provided by α-HA staining. Data from 12 image stack per condition; genotype (Number of cells Fed): Ctrl(3307), LIPA^−/−(1996), GAA^−/−(1401), NPC1^−/−(1211), NPC2^−/−(1629). Error bars depict S.D. (I) Confocal images of NPC2^−/− cells immunostained for α-LAMP1 and α-FTH1, with lysosomes marked by Filipin staining. A single z-slice is shown. Scale bar = 2 μm. (J) 3D-SIM reconstructions of NPC1^−/− or NPC2^−/− cells immunostained with α-FTH1 and the surface volume of cholesterol-rich lysosomes marked by Filipin.

Figure 5:. Visualization of multi-lamellar membranes in NPC2^−/− lysosomes by cryo-ET.

(A) Schematic showing endocytosis of dextran and its ultimate incorporation into the lysosome in Control and NPC2^−/− cells. In Control cells, dextran endocytosis successfully delivers dextran to the lysosomal lumen via vesicle fusion. In NPC2^−/− cells with multi-lamellar membranes, successful fusion and delivery of dextran is reduced and successful fusion events result in dextran present in the limited lumenal space between the limiting lysosomal membrane and the first internal membrane. (B) Control or NPC2^−/− cells were treated with dextran conjugated with Alexa647 dye and imaged by live-cell 3D-SIM. Renderings derived from 3D-SIM reconstructions are shown below. Scale bar = 2 μm. (C) As in panel B, but cells were cultured on Galactose growth media for 24h. Scale bar = 2 μm. (D) Control cells were treated for 3 days with the NPC1 inhibitor U18666A incubated alongside NPC1^−/− cells with dextran conjugated with Alexa647 dye and imaged by live-cell 3D-SIM. Renderings derived from 3D-SIM reconstructions are shown below. Scale bar = 2 μm. (E) Schematic of the plasma-FIB and cryo-ET workflow. (F) Example lamella overviews of Control and NPC2^−/− cells under 6 h EBSS nutrient starvation conditions. Scale bar = 500 nm. (G) Example tomogram slice of multilamellar vesicles in NPC2^−/− cells. Scale bar = 200 nm. (H) Quantification of MLV-containing tomograms from Control and NPC2^−/− cells. Total number of tomograms analyzed is stated above the bar charts. (I) 3D-renderings of a segmented NPC2^−/− tomogram. Zoom-ins highlighting close proximity between MLV (orange) with mitochondria (green) and a putative lysosome (pink) are shown beneath. (J) Quantification of membrane bilayer size (left) and distance between membrane leaflets (right) across three tomograms for the cytosolic membrane (CM), the enclosed membranes (EM), and the luminal membrane (LM). Quantification of the spacing between individual membranes: CM to first EM (left), between EMs (middle), and EM to LM (right). p(*) = 0.011, 0.21; p(**) = 0.0086, 0.0052. Data based on triplicate experiments (lamellae), ordinary one-way ANOVA with multiple comparisons, alpha = 0.05. Error bars depict S.D. Abbreviations: ER = Endoplasmic reticulum; MLV = Multi-lamellar vesicle.

Figure 6:. Defects in mitochondrial cristae/OXPHOS systems in NPC2^−/− cells and amelioration by extracellular iron.

(A) Heatmap depicting log₂FC of components of different mitochondrial compartments in either Fed and EBSS-treated Control and 4KO cells. Data based on quadruplicate biological replicate nMOST measurements. (B,C) Panel B: Log₂FC values for modules within OXPHOS for 4KO cells in Fed and EBSS-treated cells based on quadruplicate replicate nMOST data. Panel C: Log₂FC of replisome sub-module abundance comparing Fed versus EBSS treated Control, LIPA^−/−, GAA^−/−, NPC1^−/−, and NPC2^−/− cells based on quadruplicate biological replicate nMOST data. (D) Abundance of heatmaps in B mapped onto structure of mitochondrial respirasome (CI, CIII₂, CIV from PDB: 5XTH). Legend shows colour panel for log₂FC values. Based on quadruplicate replicate nMOST data. (E) Heatmap for components of the MICOS-MIB complex in for 4KO cells [normalized with Control]. Data based on quadruplicate biological replicate nMOST measurements. (F) Z-projections of live-cell 3D-SIM images from Control, LIPA^−/− and NPC2^−/− cells after culturing on galactose (24 h) stained with the IMS dye PKmitoRed. Scale bar = 2 μm. (G) Line-plots of dashed lines from panel F of Control, LIPA^−/− and NPC2^−/− cells on 24 h galactose growth conditions. (H) Confocal images of Control and NPC2^−/− cells grown in glucose (Fed) or Galactose (24h) followed by immunostaining with α-HA to detect TMEM192^HA and α-FTH1 to detect Ferritin. Scale bar = 10 μm. Right panel displayed quantification of FTH1 signal overlapping with lysosome staining/cell. Data from biological quadruplicates per sample (each replicate containing 5–9 stacks); p(****) <0.0001, ordinary two-way ANOVA with multiple comparisons, alpha = 0.05. Error bars depict S.D.. (I) Z-projections of live-cell 3D-SIM images from Control and NPC2^−/− cells after culturing on Galactose (72 h) with or without FAC and stained with the IMS dye PKmitoRed. Scale bar = 2 μm. (J) Line-plots of individual mitochondria from panel I. Red asterisks indicate positions of cristae. (K) Violin plot depicting the ratio of cristae to mitochondria with and without FAC addition. Data based on 132 (72 h Gal) or 148 (72 h Gal + FAC) segmented planes of ROI-stacks from data shown in panel I; p(*) = 0.0242, unpaired t.test. (L) Log₂ β-coefficient for the indicated treatments shown for all OXPHOS subunits and individual sub-complexes. (M) Log₂FC [NPC2^−/−/Control] for the indicate protein complexes under the indicated conditions (time in Galactose with or without FAC addback). Data based on triplicate biological replicate TMTpro measurements.

Figure 7:. Proteomic analysis of NPC mutants during neurogenesis with iron addback.

(A) Schematic of experimental approach to study effect of iron supplementation during NGN₂-driven neurogenesis and downstream label-free proteomic analysis. For each time-point samples were analysed in triplicates. (B) PCA plot of LFQ data, color-coded according to day of differentiation or ±FAC. (C) Bargraph of mean log₂FC (normalized within genotype day 0) mitochondrial OXPHOS-components in presence of FAC. Black asterisks indicate statistical comparison to Control cells; red asterisks indicate comparison ± FAC within genotype. Data from triplicate replicates; NPC1^−/− E2 d0: p(*) = 0.0286; d4: p(***) = 0.0003; d8, d4+FAC, d8+FAC: p(****) <0.0001. NPC2^−/− C3 d0: p(*) = 0.0420. Ordinary two-way ANOVA with multiple comparisons, alpha = 0.05. Error bars depict S.E.M.. (D) Violin plots of log2FC (normalized to Control at day 0) N- and Q-module of Complex I. A schematic of Complex I and FeS clusters is shown in the left (based on PDB: 5XTH). Abundance comparisons over a three-week differentiation time-course ± FAC treatment are shown for each genotype (grey for -FAC, red for +FAC). Black asterisks indicate statistical comparison to Control cells; red asterisks indicate comparison within genotype. Blue asterisks indicate comparison of day 8 to 16 within genotype. Data from triplicate replicates; Control d0,4,8: p(****) <0.0001; d4,8+FAC: p(****) <0.0001; d8-16: (****) <0.0001. NPC1^−/− E2 d0: (****) <0.0001; d0+FAC: p(***) = 0.0002; all other: p(****) <0.0001. NPC2^−/− C3: p(****) <0.0001. NPC2^−/− G1 d0: p(*) = 0.0396; d0+FAC: p(**) = 0.0041; all other: p(****) <0.0001. Ordinary two-way ANOVA with multiple comparisons, alpha = 0.05. Error bars depict S.E.M.. (E) Surface renderings of FeS-associated Complex I N- and Q-modules (red) overlayed with rest of the protein complex rendered in grey (PDB: 5XTH). Violinplots of log₂FC (normalized to Control at day 0) FeS-associated proteins. Abundance comparisons over a three-week differentiation time-course ± FAC treatment are shown for each genotype (grey for -FAC, red for +FAC). Black asterisks indicate statistical comparison to Control cells; red asterisks indicate comparison within genotype ±FAC. Data from triplicate replicates; Control: p(****) <0.0001. NPC1^−/− E2 d0: (*) = 0.0489; d8+FAC: p(*) = 0.0146; all other: p(****) <0.0001. NPC2^−/− C3: p(****) <0.0001. NPC2^−/− G1: p(****) <0.0001. Ordinary two-way ANOVA with multiple comparisons, alpha = 0.05. Error bars depict S.E.M..